| Grapevine is one of the most important cultivated fruit trees in the world.Vitis vinifera is widely used in wine making,fresh food,juice making and raisins making due to their high quality and high yield.However,V.vinifera has poor resistance to stress and fungal disease such as powdery mildew.Resveratrol is a well-known phytoalexin,which can inhibit the spread of many fungal diseases.In addition,Resveratrol was believed to be beneficial to human health.China is one of the three major origin centers of grapevines which ccontains abundant wild resistant grape germplasm resources.Previous studies showed that Chineses wild V.quinquangularis ‘Danfeng-2' not only has strong disease resistance but also has highcontent of resveratrol.However,the regulation mechanism of stilbenes matobolism is not clear.In this study,the transcriptome,proteome and metabolome were combined to analyze the genetic mechanism of high content of resveratrol in ‘Danfeng-2' from the transcriptional level and protein level,and to explore the genes involved in the regulation of resveratrol synthesis.And our study would lay the foundation for further analysis of the metabolic regulation mechanism of resveratrol and provide valuable gene resources for futher breeding.The main results are as follows:1.Transcriptome results indicated that there were 19,008 expressed genes in ‘Danfeng-2' and 19,373 expressed genes in ‘Cabernet Sauvignon'.There were 54 stilbene synthase genes in ‘Danfeng-2' and 48 stilbene synthase genes in ‘Cabernet Sauvignon'.Six novel stilbene synthase transcripts were identified through assembly,annotation and expression analysis of ‘Danfeng-2' specific novel transcripts.The differentially expressed genes of ‘Danfeng-2' and ‘Cabernet Sauvignon' were identified through transcriptome sequencing of the berries at four developmental stages.GO enrichment,KEGG enrichment and Map Man analysis were further conducted to determine metabolic pathways and biological processes which the differentially expressed genes involved in.Results indicated that the phenylalanine metabolic pathway,photosynthetic pathway,cell wall component-related pathway and fatty acid metabolism pathway are the main significant differential expression pathways at the Veraison stage and the Ripe stage.Furthermore,in the phenylalanine metabolic pathway,phenylalanine ammonia lyase genes and stilbene synthase genes were highly expressed in ‘Danfeng-2' whereas chalcone synthase genes were highly expressed in ‘Cabernet Sauvignon'.In addition to genes related to stilbenes metabolism,various differentially expressed genes including biotic stress genes,resistance genes,transcription factor genes,photosynthetic genes,fatty acid metabolism genes,cell wall component-related genes and lignin genes were identified.The up-regulated genes of gene clusters methioned above were 72/116,46/70,179/150,50/1,66/62,26/91,and 14/24,respectively which indicated pathways of transcriptional regulation were enhanced in ‘Danfeng-2'.2.A total of 3,751 proteins were identified through proteome analysis including Cellular process(1,180),Developmental process(198),Growth(4),Immune system process(26),Interaction with cells and organisms(120),Localization(181),Metabolic process(269),Regulation(355),Reproduction(1),Responese to stimulus(544)and Other(227).Differentially expressed proteins in four development stages of ‘Danfeng-2' and ‘Cabernet Sauvignon' were identified using proteome analysis.Results indicated that the stilbene synthase and chalcone synthase compete for coumaryl coenzyme A in the secondary metabolic pathway.And the synthetic biological processes of the stilbenes were enhanced during the Veraison and Ripe stages in ‘Danfeng-2'.3.A total of 203 metabolites were identified through GC-MS analysis.Differential metabolites of ‘Danfeng-2' and ‘Cabernet Sauvignon' during fruit development were identified using metabolism analysis.The contents of phenylalanine and hydroxycinnamic acid,key metabolites related to stilbenes synthesis were significantly higher in ‘Danfeng-2' compared with ‘Cabernet Sauvignon'.High-performance liquid chromatography was used to determine the content of resveratrol and its derivative,piceid,in berries at the Green hard,the Before veraison,the Veraison stage and the Ripe stage of V.quinquangularis ‘Danfeng-2' and V.vinifera ‘Cabernet Sauvignon'.The content of stilbenes in berries of ‘Danfeng-2' was significantly higher than that of ‘Cabernet Sauvignon' at four developmental stages,and the content of resveratrol and piceid reached the peak at ripe stage in ‘Danfeng-2'.Besides,the contents of flavonoids and anthocyanins in ‘Cabernet Sauvignon' were significantly higher than that in ‘Danfeng-2' at four development stages.4.Further genome-wide analysis was used to identify gene families involved in the synthesis of resveratrol derivatives in the phenylalanine metabolic pathway.A total of 58 o-methyltransferase genes,77 laccase genes and 49 resveratrol glycosyltransferase genes were identified.Through phylogenetic analysis,gene structure analysis and conservation motifs analysis,we found that the members in the three gene families have functional redundancy and functional differentiation.Moreover,they also have spatiotemporal expression difference during fruit development.5.Co-expression was used to identify transcription factors which were co-expressed with stilbene synthase genes.As a result,52 candidate positive regulation transcription factor genes were identified,including six b HLH,two b ZIP,nineteen ERF,sixteen MYB,eight WRKY,and one NAC genes.Besides,three candidate negative regulation transcription factors were identified,including one b HLH,one MYB,and one b ZIP gene.Additionaly,five b HLH,one b ZIP,sixteen ERF,eight WRKY,and one NAC transcription factors were up-regualted in ‘Danfeng-2'.While only nine transcription factors up-regualted in ‘Cabernet Sauvignon'.Further yeast single hybridization experiments showed that 11 of 12 highest-ranking candidate transcription factors could bind to the promoter of the stilbene synthase gene.In addition,the function of one transcription factor Vq NAC1 which is homologous to the Arabidopsis thaliana transcription factor ATAF1 regulating the abscisic acid metabolism,was analyzed.The results showed that transient expression of Vq NAC1 in grape leaves enhanced the accumulation of stilbenes and increased the expression of abscisic acid synthase-related genes and PR1 gene.6.In addition,four candidate long non-coding RNAs in ‘Danfeng-2' that regulate the expression of stilbene synthase genes were screened through co-expression analysis of 1,112 long-chain non-coding RNAs found in transcriptome data.Moreover,two of them could up-regulate the expression of stilbene synthase genes.In summary,multi-omics analysis of differential expression in ‘Danfeng-2' and ‘Cabernet Sauvignon' with GO,KEGG enrichment and Map Man analysis revealed the regulation network of stilbene synthase gene and stilbenes accumulation.In additon,we found that the expression profiles of stilbene synthase genes were regulated by various transcription factors,and six novel stilbene synthase gene transcripts specific to ‘Danfeng-2' were identified,which improved the regulatory network of the stilbene synthase genes.Addtionly,long non-coding RNAs were proved to involve in regulating the expression of stilbene synthase genes.This study provides a scientific basis for using China wild grape germplasm resource for functional research of stilbene synthase genes and breeding in the future. |
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